Die vorliegende Arbeit befasst sich mit mechanistischen Untersuchungen der fluorolytischen Sol-Gel-Synthese von nano-Magnesiumfluorid, sowie verschiedenen Abwandlungen der bekannten Synthese zur gezielten Veränderung der Eigenschaften der erhaltenen Materialien. Es werden die drei im folgenden beschriebenen Themenbereiche behandelt: Der Verlauf der Fluorolyse von Magnesiummethoxid mit methanolischer HFLösung wird mit der 19F-NMR-Spektroskopie über sechs Monate untersucht. Es wird gezeigt, dass MgF2-Nanopartikel, Agglomerate von gestörten MgF2-Partikeln und nicht umgesetzte, adsorbierte HF-Spezies in den Solen nachweisbar sind. Erstmalig werden MAS-NMR-Experimente an Solen beschrieben. Zusätzlich wird die schrittweise Fluorolyse von MgCl2 mittels NMR-Spektroskopie und XRD untersucht. Es werden drei verschiedene Synthesewege vorgestellt, mit denen eine Veränderung von Partikel- oder Kristallitgrößen erreicht wird. Eine sequentielle Synthese ermöglicht die Vergrößerung der Agglomerate im Sol. Das Erhitzen eines Sols zum Sieden unter Rückflusskühlung führt zu einem geringfügigen Anwachsen der Kristallitgröße. Durch Solvothermal-Synthesen wird eine signifikante Vergrößerung der Kristallite erzielt. Der Einfluss verschiedener Reaktionsparameter wird untersucht. Der Einfluss von MgF2 auf die Kristallisation von amorphem TiO2 wird untersucht. Es wird gezeigt, dass die Sol-Gel-Synthese von TiO2 in Gegenwart eines MgF2-Sols (min. 5 mol%) zur Kristallisation von Rutil-TiO2 bei vergleichsweise niedrigen Temperaturen führt. Verschiedene alternative Synthesen werden vorgestellt, durch die bei vergleichbaren Bedingungen nur Anatas-TiO2 erhalten werden kann. Ein möglicher Mechanismus der Strukturinduktion wird vorgeschlagen. / The present Ph.D. thesis deals with mechanistic investigations of the fluorolytic sol-gel synthesis of nano magnesium fluoride. Furthermore, variations of the well known synthesis are introduced. The aim of these variations is to tailor the properties of the synthesized materials. The thesis covers three main chapters briefly introduced below: The course of the fluorolysis of magnesium methoxide with methanolic HF-solution will be monitored for six months using 19F NMR spectroscopy. The existence of MgF2 nanoparticles and agglomerates of disturbed MgF2 particles will be proven. It is demonstrated that hydrogen fluoride does not react immediately after the addition of HF-solution. For the first time MAS-NMR experiments of sols will be conducted. Furthermore, stepwise fluorolysis of magnesium chloride will be followed by NMR spectroscopy and X-ray diffraction. In this case no intermediates will be detected. Three different synthetic approaches capable of tailoring the crystallite and particle sizes will be presented. Using a sequential synthesis leads to increased size of the agglomerates in the sols. It will be demonstrated that heating and refluxing of a sol increases the crystallite size slightly. Solvothermal synthesis will be the last method leading to significant increase in crystallite sizes. Several synthetic parameters will be varied to identify their influence on the received crystallites. The influence of nano MgF2 on the crystallisation of amorphous TiO2 is investigated. It will be shown, that the sol-gel synthesis of TiO2 in the presence of a MgF2 sol leads to the crystallisation of the rutile polymorph of TiO2. The temperature treatment for that is comparatively low and just 5 mol% MgF2 are necessary. Furthermore, a different alternative synthesis will be introduced, that gives the anatase polymorph at the same conditions. Eventually a possible mechanism for the structural induction is proposed.

<p>Glycoconjugates are biologically important molecules with diverse functions. They consist of carbohydrates of varying size and complexity, attached to a non-sugar moiety as a lipid or a protein. Glycoconjugate structures are often very complex and their intricate biosynthetic pathways makes overexpression difficult. This renders the isolation of pure, structurally defined compounds from natural sources cumbersome. Therefore, to better address questions in glycobiology, synthetic glycoconjugates are an appealing alternative. In addition, synthetic methods allow for the preparation of non-natural glycoconjugates that can enhance the understanding of the influence of structural features on the biological responses.</p><p>In this thesis, synthetic methods for the preparation of glycoconjugates, especially glycolipid analogues, have been developed. These methods make use of solid-phase chemistry and are amenable to library synthesis of series of similar compounds. Solid-phase synthesis is a technique where the starting material of the reaction is attached to small plastic beads through a linker. This allows large excess of reagents to speed up the reactions and the sometimes difficult purifications of intermediate products are reduced to simple washings of the beads.</p><p>One problem with solid-phase synthesis is the difficulties to monitor the reactions and characterize the intermediate products. Gel-phase 19 F-NMR spectroscopy, using fluorinated linkers and protecting groups, is an excellent tool to overcome this problem and to monitor solid-phase synthesis of e.g. glycoconjugates. Two novel fluorinated linkers for the attachment of carboxylic acids have been developed and are presented in the thesis. These linkers can be cleaved with both acids of varying strengths and nucleophiles like hydroxide ions, and they are stable to glycosylation conditions. In addition, a novel filter reactor for solid-phase synthesis was designed. The reactor fits into an ordinary NMR spectrometer to facilitate the reaction monitoring with gel-phase 19 F-NMR spectroscopy.</p><p>The biological applications of the synthesized glycolipids were demonstrated in two different settings. The CD1d restricted binding of glycolipids carrying the monosaccharide α-GalNAc as carbohydrate could be detected on viable cells of mouse origin. CD1d is one of several antigen presenting molecules (the CD1 proteins) that presents lipids and glycolipids to circulating T-cells that in turn can initiate an immune response. The CD1 molecules are relatively sparsely investigated, and the method to measure glycolipid binding on viable cells, as described in the thesis, has the possibility to greatly enhance the knowledge of the structural requirements for CD1-binding.</p><p>Serine-based neoglycolipids with terminal carboxylic acids were used to prepare glycoconjugate arrays with covalent bonds to secondary amines on microtiter plates. Carbohydrate arrays have great possibilities to simplify the study of interactions between carbohydrates and e.g. proteins and microbes. The usefulness of the glycolipid arrays constructed in the thesis was illustrated with two lectins, RCA120 from Ricinus communis and BS-1 from Bandeiraea simplicifolia. Both lectins bound to the array of neoglycolipids in agreement with their respective specificity for galactosides.</p><p>Glycobiology is a large area of great interest and the methods described in this thesis can be used to answer a variety of glycoconjugaterelated biological questions.</p>

Glycoconjugates are biologically important molecules with diverse functions. They consist of carbohydrates of varying size and complexity, attached to a non-sugar moiety as a lipid or a protein. Glycoconjugate structures are often very complex and their intricate biosynthetic pathways makes overexpression difficult. This renders the isolation of pure, structurally defined compounds from natural sources cumbersome. Therefore, to better address questions in glycobiology, synthetic glycoconjugates are an appealing alternative. In addition, synthetic methods allow for the preparation of non-natural glycoconjugates that can enhance the understanding of the influence of structural features on the biological responses. In this thesis, synthetic methods for the preparation of glycoconjugates, especially glycolipid analogues, have been developed. These methods make use of solid-phase chemistry and are amenable to library synthesis of series of similar compounds. Solid-phase synthesis is a technique where the starting material of the reaction is attached to small plastic beads through a linker. This allows large excess of reagents to speed up the reactions and the sometimes difficult purifications of intermediate products are reduced to simple washings of the beads. One problem with solid-phase synthesis is the difficulties to monitor the reactions and characterize the intermediate products. Gel-phase 19 F-NMR spectroscopy, using fluorinated linkers and protecting groups, is an excellent tool to overcome this problem and to monitor solid-phase synthesis of e.g. glycoconjugates. Two novel fluorinated linkers for the attachment of carboxylic acids have been developed and are presented in the thesis. These linkers can be cleaved with both acids of varying strengths and nucleophiles like hydroxide ions, and they are stable to glycosylation conditions. In addition, a novel filter reactor for solid-phase synthesis was designed. The reactor fits into an ordinary NMR spectrometer to facilitate the reaction monitoring with gel-phase 19 F-NMR spectroscopy. The biological applications of the synthesized glycolipids were demonstrated in two different settings. The CD1d restricted binding of glycolipids carrying the monosaccharide α-GalNAc as carbohydrate could be detected on viable cells of mouse origin. CD1d is one of several antigen presenting molecules (the CD1 proteins) that presents lipids and glycolipids to circulating T-cells that in turn can initiate an immune response. The CD1 molecules are relatively sparsely investigated, and the method to measure glycolipid binding on viable cells, as described in the thesis, has the possibility to greatly enhance the knowledge of the structural requirements for CD1-binding. Serine-based neoglycolipids with terminal carboxylic acids were used to prepare glycoconjugate arrays with covalent bonds to secondary amines on microtiter plates. Carbohydrate arrays have great possibilities to simplify the study of interactions between carbohydrates and e.g. proteins and microbes. The usefulness of the glycolipid arrays constructed in the thesis was illustrated with two lectins, RCA120 from Ricinus communis and BS-1 from Bandeiraea simplicifolia. Both lectins bound to the array of neoglycolipids in agreement with their respective specificity for galactosides. Glycobiology is a large area of great interest and the methods described in this thesis can be used to answer a variety of glycoconjugaterelated biological questions.

The development of molecular receptors for anion recognition has become an important aspect of supramolecular chemistry. In this thesis, we focused our attention to the study of systems for fluoride recognition in water. Fluoride is indeed an anion of interest due to its implication in environmental and health related issues. Furthermore, its small size and high hydration energy make its recognition in water particularly challenging.<p>Most of the synthetic systems reported for fluoride recognition have been extensively studied in organic solvents (DMSO, acetonitrile) using tetrabutylammonium fluoride (TBAF) as the source of fluoride. In many cases, titra- tion behaviours are observed that cannot be ascribed to a classical 1:1 binding isotherm, deprotonation problems of Brønsted-Lowry acid type of receptors aside. In the first part of our thesis we investigated, using a uranyl-salophen re- ceptor which recognizes fluoride via Lewis Acide/Base interactions, the origin of the unusual titration behaviour. Via UV/vis, 19F and 1H NMR spectroscopies, we have been able to highlight that the equilibrium between the fluoride and the corresponding bihalide ion, HF−2 ,which is inevitably generated along with the hydroxide anion in situ due to trace amounts of water, can be at the ori- gin of this singular behaviour. Our results put to light that when undertaking titrations with fluoride in DMSO, the fluoride–bihalide equilibrium can affect the data and that the latter species can even be the dominating species at low TBAF concentrations. When varying the solvent from DMSO to acetonitrile, the s-shape titration curves observed by UV/vis are no longer observed for the uranyl-salophene receptor that we studied. The fluoride-bifluoride equilibrium is still present but both of the anions generated in this process are recognised by the uranyl-salophene receptor with similar affinity constants above 10^6 M−1.<p>The second part of our work was devoted to finding ways to solubilize anion receptors that are efficient in organic solvents, into an aqueous environment. Two approaches were investigated: (i) grafting of the receptors onto silica nanoparticles and (ii) the micellar incorporation of the receptors. For the first strategy, we developed two silylated urea-based receptors. These receptors were first studied in organic solvents (DMSO and/or acetonitrile) where they showed selectivity, among halides, towards fluoride. Once grafted on the silica nanoparticles, due to the fact that hydroxyl groups and solvent molecules are present in the silica matrix, fluoride recognition was not possible.<p>We explored it with different simple H-bond based urea receptors in the second srategy. With the cationic surfactants, cetyl trimethyl ammonium chloride and bromide, the counter-ions of the micelles interfere with the fluoride recognition. With the neutral surfactant triton X-100, the incorporation of the anion receptors proved to be difficult. Moreover, the variations observed in the UV/vis spectra upon titrations were too small to be able to make any conclusions about fluoride recognition. / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19003号 / 工博第4045号 / 新制||工||1622(附属図書館) / 31954 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 白川 昌宏, 教授 佐藤 啓文, 教授 梶 弘典 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

<p>An efficient and versatile non-destructive method to analyze the progress of solid-phase glycoconjugate synthesis with gel-phase ¹⁹F NMR spectroscopy is described. The method relies on use of fluorinated linkers and building blocks carrying fluorinated protective groups. Commercially available fluorinated reagents have been utilized to attach the protective groups. </p><p>The influence of resin structures for seven commercial resins upon resolution of gel-phase ¹⁹F NMR spectra was investigated. Two different linkers for oligosaccharide synthesis were also developed and successfully employed in preparation of α-Gal trisaccharides and a n-pentenyl glycoside. Finally, reaction conditions for solid-phase peptide glycosylations were established.</p>

An efficient and versatile non-destructive method to analyze the progress of solid-phase glycoconjugate synthesis with gel-phase 19F NMR spectroscopy is described. The method relies on use of fluorinated linkers and building blocks carrying fluorinated protective groups. Commercially available fluorinated reagents have been utilized to attach the protective groups. The influence of resin structures for seven commercial resins upon resolution of gel-phase 19F NMR spectra was investigated. Two different linkers for oligosaccharide synthesis were also developed and successfully employed in preparation of α-Gal trisaccharides and a n-pentenyl glycoside. Finally, reaction conditions for solid-phase peptide glycosylations were established.

In the first part of the thesis, protein-ligand interactions were investigated using the chaperone LcrH, from Yersinia as target protein. The structure of a peptide encompassing the amphipathic domain (residue 278-300) of the protein YopD from Yersinia was determined by NMR in 40% TFE. The structure of YopD278-300 is a well defined α-helix with a β-turn at the C-terminus of the helix capping the structure. This turn is crucial for the structure as peptides lacking the residues involved in the turn are unstructured. NMR relaxation indicates that the peptide is not monomeric. This is supported by intermolecular NOEs found from residue Phe280 to Ile288 and Val292 indicative of a multimeric structure with the helical structures oriented in an antiparallel manner with hydrophobic residues forming the oligomer. The interaction with the chaperone LcrH was confirmed by 1H relaxation experiments and induced chemical shift changes in the peptide Protein-ligand interactions were investigated further in the second paper using a different approach. A wide range of substances were used in screening for affinity against the chaperones PapD and FimC from uropathogenic Escherichia coli using 1H relaxation NMR experiments, surface plasmon resonance and 19F NMR. Fluorine NMR proved to be advantageous as compared to proton NMR as it is straight forward to identify binding ligands due to the well resolved 19F NMR spectra. Several compounds were found to interact with PapD and FimC through induced line-broadening and chemical shift changes for the ligands. Data corroborate well with surface plasmon resonance and proton NMR experiments. However, our results indicate the substances used in this study to have poor specificity for PapD and FimC as the induced chemical shift is minor and hardly no competitive binding is observed. Paper III and IV is an investigation of the structural features of the allergenic 2S albumin Ber e 1 from Brazil nut. Ber e 1 is a 2S albumin previously identified as the major allergen of Brazil nut. Recent studies have demonstrated that endogenous Brazil nut lipids are required for an immune response to occur in vivo. The structure was obtained from 3D heteronuclear NMR experiments followed by simulated annealing using the software ARIA. Interestingly, the common fold of the 2S albumin family, described as a right-handed super helix with the core composed of a helix bundle, is not found in Ber e 1. Instead the C-terminal region is participating in the formation of the core between helix 3, 4 and 5. The dynamic properties of Ber e 1 were investigated using 15N relaxation experiments and data was analyzed using the model-free approach. The analysis showed that a few residues in the loop between helix 2 and 3 experience decreased mobility, compared to the rest of the loop. This is consistent with NOE data as long range NOEs were found from the loop to the core region of the protein. The anchoring of this loop is a unique feature of Ber e 1, as it is not found in any other structures of 2S albumins. Chemical shift mapping of Ber e 1 upon the addition of lipid extract from Brazil nut identified 4 regions in the protein where chemical shift perturbations were detected. Interestingly, all four structural clusters align along a cleft in the structure formed by helix 1-3 on one side and helix 4-5 on the other. This cleft is big enough to encompass a lipid molecule. It is therefore tempting to speculate whether this cleft is the lipid binding epitope in Ber e 1.

Mucin is a term used to describe a large family of heavily glycosylated proteins which are present on the surfaces of secretory epithelial cells and are overexpressed by many carcinomas. Membrane-bound mucin MUC1 is of special interest. Its backbone consists of repeating units of twenty amino acids with five potential glycosylation sites. These sites are expanded to structures like the T (Galβ(1-&gt;3)GalNAcα-Ser/Thr) and Tn (GalNAcα-Ser/Thr) antigens by the action of various glycosyltransferases. In different types of carcinomas these epitopes are being terminated by sialic acid residues to form among others: 2,3-sialyl-T and sialyl-Tn structures due to the elevated levels of different sialyltransferases. Solid-phase synthesis of the selected antigens derived from the mucin MUC1 has been developed and optimized. A chemoenzymatic approach has been used to effectively prepare 2,3-sialyl-T and 2,6-sialyl-Tn glycopeptides. The formation of intramolecular sialic acid lactones in presence of acetic acid was investigated. The stability of lactones formed from 2,3-sialyl-T towards water was studied using NMR spectroscopy and it appeared that 1''-&gt;2' lactone displayed remarkable strength to hydrolysis and it was suggested as a candidate for cancer vaccine. Gel-phase 19F NMR spectroscopy is known to be a very good tool to characterize resin-bound products using fluorinated protecting groups and linker molecules. The hydrophobic peptide LLLLTVLTV, which is a fragment from the MUC1 signal sequence, was prepared using solid-phase synthesis according to a modified Fmoc protocol with more active coupling reagent, stronger base, and the isopropylidene dipeptide Fmoc-Leu-Thr-(ΨMe,Mepro)-OH. Gel-phase 19F NMR spectroscopy was used to evaluate peptide chain aggregation and coupling and deprotection efficiency. A carbamate linker strategy proved to be effective in solid-phase synthesis of serine-based neoglycolipids with terminal amino functionality. Neoglycolipids were covalently bound to secondary amines in microtiter plates using squaric acid ester methodology. These arrays have potential to study the interactions between carbohydrates and e.g. proteins and microbes. The new fluorinated α-amino protective group [1-(4-(4-fluorophenyl)-2,6-dioxocyclohexylidene)ethyl] Fde was developed. This group is cleaved with hydrazine in DMF solution. By using amino acids protected with this group, it was possible to quantify the efficiency of peptide coupling using gel-phase 19F NMR spectroscopy.

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